Despite the success of the Back-to-Sleep campaign, the sudden infant death syndrome (SIDS) remains the leading cause of postneonatal infant mortality in the United States today. The major discovery of this program project in the last cycle was that multiple developmental abnormalities in the medullary serotonergic (5-HT) system are associated with SIDS. These human findings in Project 1 have lead to the establishment of a critical role for the medullary 5-HT system in the state-related regulation of multiple homeostatic functions in animal models in Projects 2-4. Additionally, they have lead to establishment of the developmental profile at the molecular level of the brainstem 5-HT system in Project 5. These discoveries have lead to the following over-riding hypothesis for the proposed third cycle: an important subset of SIDS is due to a developmental disorder of the medullary 5-HT system and related neuromodulator systems that are incurred prenatally, but which exerts its effects postnatally- during the first 6 months of life when the newborn, in transitioning to extrauterine life, undergoes maturation of circuitry for maintaining independent homeostasis and is at the greatest risk for SIDS. This disorder impairs protective homeostatic responses to potentially life-threatening, but often occurring, stressors during infant sleep that lead in turn to homeostatic derangements (hypoxia, hypercarbia), each potentially non-lethal in themselves, but which in combination precipitate death. Our objectives are: 1) To define the role of multiple transmitters/modulators in the neurochemical medullary pathology in SIDS infants;2) To determine in rodent models the effect of homeostatic stressors-alone and in combination-upon autonomic and respiratory function during different sleep states, in males and females, and at different ages;3) To determine the role of chronic intermittent hypoxia in the potentiation of the brainstem pathology in SIDS;4) To determine the role of y-aminobutyric acid (GABA), orexin, substance P, norepinephrine, and acetylcholine in interacting with the medullary 5-HT system in homeostatic regulation in animal models;5) To establish the organization and connectivity of the medullary 5-HT system-the "integrator" of diverse homeostatic functions mediated by multiple effector neurons;6) To inhibit ("silence") molecularly defined subsets of 5-HT and GABA neurons and determine the physiological consequences in the living mouse. In the proposed third cycle, we will test the over-riding hypothesis utilizing a multidisciplinary approach in which human, animal, tissue slice, cell culture, and developmental data are integrated together, thereby informing and expanding upon each other towards establishing the pathogenesis of SIDS from the cellular to systems level. PROJECT 1: NEUROCHEMICAL PATHOLOGY IN SIDS BRAINSTEMS (Kinney, Hannah, M.D., pp 163-206) DESCRIPTION: The major discovery of Project 1 in the last cycle was that multiple abnormalities in the medullary serotonergic (5-HT) system are associated with SIDS. We now envision SIDS as a complex and heterogeneous process which involves multiple transmitters in addition to 5-HT, multiple stressors acting simultaneously, and multiple genetic and environmental factors, including chronic hypoxia and male gender, augmenting the brainstem defects. Our Specific Aims are: 1) To determine the neurochemical anatomy of Y-aminobutyric acid (GABA) in the medulla in SIDS cases. We will determine the number and density of GABA neurons, percent of 5-HT neurons that co-localize with GABA, GABAA receptor binding, and cellular localization of GABAA receptor subunits in SIDS cases compared with controls adjusted for age. 2) To determine the neurochemical anatomy of SP-expressing neurons and NK1 receptors in the medulla in SIDS cases. We will determine the number and density of SP neurons, the percent of 5-HT neurons that co-localize SP, SP binding, and cellular localization of NK1 in SIDS cases compared to controls adjusted for age. We will also determine if the SIDS cases with 5-HT1A and putative GABAA receptor binding abnormalities are the same as those with SP binding abnormalities. 3) To determine the role of estrogen and testosterone in the medullary pathology of SIDS. We will test the hypotheses that: androgen receptor binding is elevated in SIDS cases (male and female) in the medullary 5-HT system and/or its projection sites compared to controls adjusted for age;high serum testosterone levels correlate with high androgen binding in the same SIDS cases;and male SIDS cases with high serum testosterone levels have lower 5-HT1A receptor binding than SIDS infants with low serum testosterone levels and controls. 4) To determine the role of chronic intermittent hypoxia in the medullary pathology in SIDS and the role of compromised responses to hypoxia due to a defect in 5-HTmediated plasticity. We will test the hypothesis that SIDS cases show a reduced hypoxic response in the hypoglossal nucleus compared to hypoxic cases, as reflected in reduced hypoxic markers, e.g., erythropoietin. This hypothesis is based on the idea that hypoxic cases are equipped with a "normal" ability to respond to hypoxia, whereas the SIDS cases have an impaired ability. We will then test the hypothesis that markers of 5-HT-mediated plasticity to intermittent hypoxia (BDNF, TrkB receptors) are reduced in SIDS cases compared to hypoxic cases in the hypoglossal nucleus. This finding would suggest an inability of the SIDS infants to adapt to intermittent hypoxia due to 5-HT raphe-related abnormalities and impaired long-term facilitation. The proposed studies build upon an emerging picture of a complex brainstem phenotype in SIDS based upon a unique database accrued over 20 years in our laboratory.